Identification of Proteins Affecting Assembly, Regulation, and Function of Thylakoid Protein Complexes

本研究项目将利用蓝细菌集胞藻PCC 6803的基因组序列（可通过http：//www.kazusa.or.jp/cyano/cyano.html获得），从功能上鉴定参与催化该蓝细菌类囊体膜中电子转移的蛋白复合物的调节、组装和功能的“新”蛋白。该项目涉及对自发突变体的正选择（1）其中光系统II活性降低或消除，或（2）其中特定基因的表达调节被修改。通过筛选缺乏光系统I的集胞藻属PCC 6803菌株的高光耐受性进行阳性选择;该菌株对强光敏感，但如果光系统II受损，或者如果类囊体膜中电子传递过程的速率或特定氧化还原组分的调节已经改变，则该菌株可以在高光强度下繁殖。集胞藻PCC 6803提供了一个强大的方法来快速识别自发突变的位置，导致高光耐受的光系统I-少的菌株。来自这些阳性选择的突变体的DNA将通过限制性内切酶进行片段化;将在凝胶上对每个消化物（用一种酶处理）进行大小分离，并检测凝胶切片（每个切片反映一定大小范围的DNA）是否与衍生自发突变体的原始（高光敏）菌株具有功能互补。一个完整的限制性内切酶图谱的集胞藻属PCC 6803基因组已经构建了16个酶，削减该蓝藻基因组300-1000倍。对于16种限制性内切酶中的每一种，通过其基因组位置鉴定的所得片段已按大小排序并汇编成表。基于可与每种限制性酶功能互补的片段的大小范围，可从每个表中选择限制性片段的适当大小范围。将这些限制性片段（每个通过它们在基因组上的位置鉴定）合并，然后根据每个片段末端的基因组核苷酸编号进行分选。在检查该排序列表时，可以鉴定核苷酸编号的簇。通常只能找到一个单一的簇，其中每个酶的每个选定大小区域都有一个代表。通过鉴定该簇中所有序列之间共有的区域，可以确定携带感兴趣的突变（通常不长于2kbp）的结构域。通过PCR扩增目的结构域，确定突变，从而可以简单地定位和鉴定未知基因中的突变，甚至不需要克隆。一些互补的二级突变可能在已知基因中，但其他突变将在开放阅读框中，其功能尚不清楚。这些开放的阅读框架将是特别感兴趣的，因为获得的结果将有助于将作用分配给这些具有未知功能的已知序列。这种方法已经导致了一个开放的阅读框架，这是类似的叶绿体ycf 39和影响突变体中的光系统II功能的鉴定。该项目的下一部分将是对已确定的开放阅读框架的作用进行初步分析。分析的类型将取决于开放阅读框和突变体的表型，但可能涉及以下内容：（1）序列同源性搜索，（2）电子传递的功能分析，（3）基因表达研究，和（4）蛋白质合成和降解测量。这项工作将导致鉴定目前未知的基因，其产物可能参与光系统II组装，在电子转移出类囊体膜中的质体醌库，或在控制细胞中的调节过程中。这是一个开放的领域，但现在集胞藻PCC 6803的基因组测序已经完成，可以进行有效和全面的分析，如本文所述。这一研究领域不仅是大规模测序工作的重要补充，因为它提供了有关开放阅读框架功能的信息，而且有望为类囊体膜中电子传递所需的调控机制和组装过程提供新的见解。

This research project will utilize the genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803 (accessible through http://www.kazusa.or.jp/cyano/cyano.html) to functionally identify "new" proteins involved in the regulation, assembly and function of protein complexes that catalyze electron transfer in thylakoid membranes of this cyanobacterium. This project involves positive selection for spontaneous mutants (1) in which photosystem II activity is decreased or abolished, or (2) in which regulation of expression of particular genes has been modified. Positive selection will occur by screening for high-light tolerance of the Synechocystis sp. PCC 6803 strain that lacks photosystem I; this strain is sensitive to high light but can be propagated at high light intensity if photosystem II is impaired or if rates of electron transport processes in the thylakoid membrane or the regulation by particular redox components have been altered.The availability of the genomic DNA sequence of Synechocystis sp. PCC 6803 provides a powerful approach to a rapid identification of the location of spontaneous mutations that lead to high-light tolerance of the photosystem I-less strain. DNA from these positively selected mutants will be fragmented by restriction enzymes; each digest (treated with one enzyme) will be size-separated on a gel and gel slices (each reflecting a certain size range of DNA) will be tested for functional complementation of the original (high-light sensitive) strain from which the spontaneous mutant was derived. A complete restriction map of the Synechocystis sp. PCC 6803 genome has been constructed for 16 enzymes that cut this cyanobacterial genome 300-1000 times each. For each of the 16 restriction enzymes, the resulting fragments, identified through their genomic position, have been sorted by size and compiled into a table. Based on the size range of the fragments that can functionally complement for each restriction enzyme, an appropriate size range of restriction fragments can be selected from each table. These restriction fragments (each identified through their position on the genome) are pooled, and are then sorted according to the genomic nucleotide number at the end of each fragment. Upon inspection of this sorted list, clusters of nucleotide numbers can be identified. Generally only a single cluster can be found in which there is one representative from each of the selected size regions for each of the enzymes. By identifying the region that is common between all sequences in this cluster, the domain carrying the mutation of interest (generally not longer than 2 kbp) can be determined. The domain of interest will be amplified by PCR, the mutation will be determined, and mutations in unknown genes can thus be simply mapped and identified, even without cloning.Some of the complementing secondary mutations may be in known genes, but others will be in open reading frames for which no function is known yet. These open reading frames will be of particular interest as the results obtained will help to assign a role to these known sequences with unknown function. This method already has led to the identification of an open reading frame that is similar to ycf39 from chloroplasts and that affects photosystem II function in mutants. The next part of the project will be the preliminary analysis of the role of open reading frames that were identified. The types of analysis will depend on the open reading frame and on the phenotype of the mutant, but may involve the following: (1) sequence homology searches, (2) functional analysis of electron transport, (3) gene expression studies, and (4) protein synthesis and degradation measurements.This work will lead to the identification of currently unknown genes whose products may be involved in photosystem II assembly, in electron transfer out of the plastoquinone pool in thylakoid membranes, or in control of regulatory processes in the cell. This is an open field, but rapid progress will be made now the genomic sequencing of Synechocystis sp. PCC 6803 has been completed, enabling effective and comprehensive means of analysis such as the one described here. This area of research not only is an important complement to large-scale sequencing efforts, in that it provides information regarding the function of open reading frames, but also is expected to provide novel insights into the regulation mechanisms and assembly processes required for electron transport in thylakoid membranes.